International cancer genome characterization efforts have identified several protein phosphatase 2A (PP2A) subunits as recurrently mutated or deleted in human cancers at a higher frequency than originally anticipated. This observation is not surprising since PP2A, the major serine-threonine phosphatase family in human cells, is broadly important in controlling cell signaling and homeostasis and particular PP2A subunits have been shown to act as tumor suppressor genes. However, progress in understanding PP2A has been confounded by the fact that the enzyme exists in at least 80 different isoforms. The study of the interactions of transforming proteins encoded by DNA tumor viruses and host cell proteins has identified many mechanisms that also contribute to the genesis of spontaneously arising cancers. In particular, the SV40 Early Region proteins bind to and inactivate several tumor suppressor gene products including retinoblastoma (RB) and p53. Studies of both SV40 small t antigen (SV40ST) and PyST, which interact with PP2A, have allowed us to begin to see how particular forms of PP2A contribute to tumor suppression. We have previously shown that suppressing particular PP2A subunits phenocopies cell transformation induced by SV40 ST and that these same PP2A subunits are tumor suppressor genes in human cancers. Using newly developed genome scale tools, we have systematically identified the specific PP2A subunits that contribute to cell transformation and have shown that loss of these subunits perturbs three pathways essential for cell transformation (WNT, MYC, AKT). Although these studies have advanced our understanding of how perturbation of PP2A by SV40ST contributes to transformation, we lack a detailed molecular understanding of the molecular connections between PP2A and the pathways essential for cell transformation. We propose to integrate innovative genetic and proteomic technologies to identify substrates and pathways perturbed by tumor suppressor PP2A subunits. In recent work we have found that the STRiatin Interacting Phosphatase And Kinase (STRIPAK) complex not only binds ST-PP2A complexes but is also required for cell transformation induced by altering PP2A function. We hypothesize that ST, or mutations in tumor suppressor PP2A subunits, reprogram the PP2A holoenzyme complex not only by displacing tumor suppressor B subunits but also by modulating the interaction of PP2A with the STRIPAK complex in cancers of both viral and non-viral etiology. In this Project we will use biochemical, genetic and cell biological approaches to understand how PP2A-STRIPAK interactions induce cell transformation to not only enhance our mechanistic understanding of this tumor suppressor family but will also provide new insights into the pathways that help program the malignant state. In addition, these studies will provide a foundation for strategies to target these pathways therapeutically.